Russian Journal of Genetics, Vol. 41, No. 3, 2005, pp. 256–261. Translated from Genetika, Vol. 41, No. 3, 2005, pp. 334–340. Original Russian Text Copyright © 2005 by Yurenkova, Kubrak, Titok, Khotyljova.
PLANT GENETICS
Flax Species Polymorphism for Isozyme and Metabolic Markers S. I. Yurenkova, S. V. Kubrak, V. V. Titok, and L. V. Khotyljova Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, 220072 Belarus; fax: (172)84-19-17; e-mail:
[email protected] Received December 15, 2003
Abstract—Genetic diversity of flax isozyme patterns of 6-phosphogluconate dehydrogenase, glutamate-oxaloacetate transaminase, and cytochrome-c-oxidase in leaves, as well as the level and the relative amounts of fatty acids in seed oil were studied in flax species. The isozyme loci examined were found to be polymorphic. It was shown that each of the flax species studied was characterized by strictly defined amounts and proportions of fatty acids, i.e., the fatty acid composition of the seed oil may be used as a species diagnostic trait. Our results suggest that all of the flax species studied have the same ancestor. Linum grandiflorum was shown to be a phylogenetic branch split at an early stage of the flax evolution.
INTRODUCTION According to a number of authors, the genus Linum L. includes more than 20 species [1, 2]. Owing to its cultivation at different latitudes, this genus is fairly polymorphic, thus having a complicated and unclear taxonomy. In the middle of the 20th century, the systematics of the genus Linum was developed on the basis of the geographical principle of morphological differences, which are not always taxonomically valuable, and the ability to yield fertile hybrid progeny [2, 3]. At present, the evolutionary and systematic relationships and genetic divergence within this genus have been clarified using cytological and genetic methods, which include such modern techniques as C-banding of chromosomes, RAPD–PCR analysis, electrophoretic fractionation of seed storage proteins, gas chromatography for determining fatty acid composition of seed oil, etc. [1, 2, 4, 5]. Owing to clear and stable isozyme patterns, isozyme analysis as the most available and generally accepted approach is used to modify and expand the traditional methods based on classical genetic markers [6]. Isozymes have been widely applied in plant gene pool studies as markers of genetic variation. However, analysis of polymorphism studies of various isozyme systems among the members of the Linum showed that this line of research has been yet insufficiently investigated. The results of studies in this direction will provide information on inter- and intraspecific genetic variation, evaluate the mechanisms of selection and evolution processes in Linum populations, and verify interspecific relationships within this genus. The aim of the present study was estimating genetic polymorphism of various flax species by comparative analysis of isozyme patterns of 6-phosphogluconate dehydrogenase, glutamate-oxaloacetate transaminase,
and cytochrome-c-oxidase in plant leaves to detect loci and alleles controlling the isozyme synthesis, as well as analyzing the fatty acid composition of seed oil as a metabolic parameter. MATERIALS AND METHODS We used the following wild Linum species: flowering flax L. grandiflorum Desf., 2n = 16; pale flax L. bienne Mill., 2n = 30; blue flax L. perenne L., 2n =18; L. austriacum L., 2n = 18; and two subspecies of cultivated flax L. usitatissimum L.: fiber flax L. usitatissimum L. subsp. usitatissimum convar. elongatum, cultivar Svetoch, 2n = 30 and intermediate flax L. usitatissimum L. subsp. usitatissimum convar. usitatissimum, cultivar Koto, 2n = 30, according to the classification in [7]. Polyacrylamide gel (7%) was prepared according to Davis [8]. For electrophoresis of cytochrome-c-oxidase and glutamate-oxaloacetate transaminase, green leaves of plants at the blossoming phase were used; for 6-phophogluconate dehydrogenase, we used greed leaves of seedlings. Green seedlings were grown in a photo container using luminescent lamps LB-40 and illumination regime 14/10 h (day/night) at 25°C. To prepare enzyme extracts, the plant material was squashed in a Tris-glycine buffer, pH 8.3, containing 0.005 M EDTA and 0.01 M β-mercaptoethanol. After electrophoresis, gels were stained using standard procedures adapted for flax [9]. The results are presented in Figs. 2–4 as zymograms. For more precise description of the results, the isozyme bands are characterized by relative electrophoretic mobilities (Rf ), which are given on the scale at the left side of each figure. Fatty acids were extracted and identified using a procedure by Welch [10] with our modifications. Ground seeds (6 to 8 mg) were placed in a 2% sulfuric acid solution in absolute methanol containing margaric
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Detector sign
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MEFA: C10:0, capric C12:0, lauric C14:0, myristic C14:1, myristoleic C15:0, pentadecanoic C16:0, palmitic C16:1, palmitoleic C17:0, margaric C18:0, stearic C18:1, cis-9-oleic C18:1, elaidic C18:2, linoleic C18:3, α-linolenic C20:0, arachidic C20:1, eicosenoic; C20:2, eicosadienoic C20:3, eicosatrienoic C22:0, behenic C22:1, erucic
12.934 C18:3
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Fig. 1. Chromatogram of fractionation of fatty acid methyl esters from seed oil of L. perenne L.
acid (C17:0; 0.27 mg/ml, intrinsic standard). The samples were hydrolyzed in sealed ampoules at 80°C to obtain methyl ethers of fatty acids [11]. These were extracted with hexane and fractionated by liquid-gas chromatography in a Hewlett–Packard 4890D unit equipped with plasma ionizing detector and capillary column HP-Innowax 0.32 mm × 30 m with a 0.5-µm carrier. Analysis was conducted at helium flow of 26 cm/s, column temperature of 200°C, and injector and detector temperature of 250°C. The volume of the sample was 1 µl. Individual fatty acids were identified according to the retention time in separation of their standard mixtures (Supelco Park, United States) and estimated in percent of the total weight in relation to the intrinsic standard (Fig. 1). The iodine value was estimated taking into the account the sample contents of palmitoleic, oleic, linoleic, α-linolenic, eicosenoic, and erucic acids [12]. The table lists mean values averaged over three analytic replications. RESULTS AND DISCUSSION Comparative analysis of electrophoretic fractionation of 6-phosphogluconate dehydrogenase (6-PGD, E.C. 1.1.1.44) in green leaves of plants in different flax species revealed polymorphism of the enzyme patterns, which was related to the absence of some zymogram components and change in their relative activity or RUSSIAN JOURNAL OF GENETICS
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electrophoretic mobility (Fig. 2a). The 6-PGD spectrum in leaves of fiber flax and intermediate flax at the green seedling stage was represented by three isoforms (bands with Rf 0.48 and 0.50 are intensely stained, band with Rf 0.45 is low active), whose formation is controlled by the Pgd-1 allele of locus PGD-1 [13]. Zymograms of L. bienne also showed three isozymes. However, these 6-PGD isozymes differed from those of fiber flax in the intensity of their expression in gel: bands with Rf 0.45 and 0.48 were intensely stained, while Rf 0.5 fraction was represented by a minor isoform (Fig. 2a). Apparently, synthesis of these isozymes is controlled by allele Pgd-11 of the PGD-1 locus, which is characterized by the fraction activity presented above. Patterns of 6-PGD in L. perenne and L. austriacum consisted of four bands. Three fast bands had the same electrophoretic mobility and staining intensity as those of L. bienne, which suggests the presence of the Pgd-11 allele in their genome. The fourth component manifested itself by a highly active slow band (Rf 0.32). This may indicate that two PGD-controlling loci function in L. perenne and L. austriacum at the green seedling stage: PGD-1, in this case allele Pgd-11 determining synthesis of three fast 6-PGD fractions, and PGD-2, controlling the slow isoform of this enzyme. Note that in fiber flax and intermediate flax, locus PGD-2 is also active at early stages of development (Fig. 2b). Studies carried out on collections of fiber flax and intermediate 2005
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Fig. 2. Schematic representation of zymograms of 6-phosphogluconate dehydrogenase isozymes of (a) flax species (leaves of 30day green seedlings): 1, fiber flax, cultivar Svetoch; 2, intermediate flax, cultivar Koto; 3, L. bienne; 4, L. austriacum; 5, L. perenne; 6, L. grandiflorum and (b) cultivars of fiber flax and intermediate flax (cotyledon leaves of 4-day etiolated seedlings): 1, Belinka; 2, Viking; 3, Hilda; 4, Orshanskii-2; 5, Dashkovskii; 6, Svetoch; 7, Leorkovskii; 8, L-41; 9, Vander; 10, Fibra; 11, Koto; 12, Baltuchyai; 13, K-6307; 14, Laser; 15, Mogilevskii-1.
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Fig. 3. Schematic representation of zymograms of glutamateoxaloacetate transaminase of the flax species (leaves of plants at the blossoming stage): 1, fiber flax, cultivar Svetoch; 2, intermediate flax, cultivar Koto; 3, L. bienne; 4, L. austriacum; 5, L. perenne; 6, L. grandiflorum.
Fig. 4. Schematic representation of zymograms of cytochrome-c-oxidase (a) of the flax species (leaves of plants at the blossoming stage): 1, fiber flax, cultivar Svetoch; 2, intermediate flax, cultivar Koto; 3, L. bienne; 4, L. austriacum; 5, L. perenne; 6, L. grandiflorum and (b) cultivars of fiber flax (cotyledon leaves of 4-day etiolated seedlings).
flax have revealed two or three isozymes of slow 6PGD in leaves of etiolated seedlings, depending on the genotype analyzed. This indicates, on the one hand, active functioning of the PGD-2 locus at this developmental stage, and, on the other hand, the presence of
several alleles at this locus. At later time of plant development (green seedling stage), this locus is inactivated in fiber flax (Fig. 2a) [13]. The pattern of L. grandiflorum at the green seedling stage consists of three components. The slow part of the 6-PGD zymograms of this
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Content of fatty acids (mass % of their sum), their sum (Σ, µg/mg crude weight), and iodine value (IN, relative units) in seeds of the flax species Fatty acids C10:0 C12:0 C14:0 C14:1n-9 C15:0 C16:0 C16:1n-9 C18:0 C18:1n-9-cis C18:1n-9-trans C18:2n-6 C18:3n-3 C20:0 C20:1n-9 C20:2n-6 C20:3n-3 C22:0 C22:1n-9 Σ IN
Flax species fiber flax
intermediate flax L. grandiflorum
L. austriacum
L. perenne
L. bienne
0.05 ± 0.00 ND* 0.15 ± 0.01 0.06 ± 0.00 0.22 ± 0.01 5.70 ± 0.39 0.04 ± 0.00 4.67 ± 0.19 28.03 ± 2.11 0.47 ± 0.03 13.53 ± 1.14 46.34 ± 3.23 0.22 ± 0.01 0.24 ± 0.02 ND ND 0.28 ± 0.03 ND
0.05 ± 0.00 ND 0.12 ± 0.01 0.06 ± 0.00 0.19 ± 0.02 6.12 ± 0.30 0.06 ± 0.00 3.47 ± 0.31 16.94 ± 1.17 0.64 ± 0.03 17.52 ± 1.25 54.19 ± 4.64 0.16 ± 0.01 0.21 ± 0.01 0.05 ± 0.00 0.08 ± 0.01 0.14 ± 0.01 ND
0.08 ± 0.01 0.08 ± 0.01 0.23 ± 0.02 0.09 ± 0.01 0.32 ± 0.04 7.71 ± 0.51 0.17 ± 0.01 6.84 ± 0.45 35.94 ± 3.42 0.81 ± 0.07 12.58 ± 1.71 34.20 ± 2.77 0.43 ± 0.03 0.36 ± 0.02 ND ND 0.16 ± 0.00 ND
0.06 ± 0.01 0.32 ± 0.04 0.26 ± 0.02 0.07 ± 0.00 0.47 ± 0.04 5.34 ± 0.33 0.05 ± 0.00 2.89 ± 0.17 25.60 ± 1.78 0.54 ± 0.07 21.03 ± 2.33 40.58 ± 5.14 1.31 ± 0.12 0.54 ± 0.04 0.29 ± 0.01 ND 0.60 ± 0.04 0.05 ± 0.00
0.05 ± 0.01 ND 0.29 ± 0.04 0.06 ± 0.00 0.41 ± 0.02 4.63 ± 0.27 0.05 ± 0.00 2.37 ± 0.21 19.26 ± 1.56 0.43 ± 0.05 23.09 ± 2.14 47.00 ± 5.01 1.13 ± 0.07 0.43 ± 0.04 0.21 ± 0.01 0.05 ± 0.00 0.50 ± 0.07 0.04 ± 0.00
0.05 ± 0.00 0.01 ± 0.00 0.09 ± 0.00 0.06 ± 0.00 0.16 ± 0.02 6.17 ± 0.47 0.04 ± 0.00 2.90 ± 0.42 16.92 ± 1.49 0.64 ± 0.03 17.40 ± 1.31 54.95 ± 4.12 0.13 ± 0.01 0.24 ± 0.01 0.03 ± 0.00 0.08 ± 0.01 0.13 ± 0.03 ND
285.60 177.1
253.76 196.0
194.17 151.8
200.26 173.1
217.57 188.5
292.61 197.8
* ND, not detected.
flax species exhibits one fraction, whose mobility is similar to the corresponding bands in L. perenne and L. austriacum, but its expression in gel is low. This may be explained by the fact that locus PGD-2, controlling the slow isoforms, includes both normal (Pgd-2) and recessive (pgd-2) alleles. In the fast part of the L. grandiflorum 6-PGD pattern, two isozymes were found, one of which coincided with that of L. perenne, L. austriacum, and L. bienne in electrophoretic mobility (Rf 0.45) and staining intensity, while the other had higher mobility (Rf 0.54) than these species (Fig. 2a). These results indicate that the loci controlling 6-PGD isozymes in flax are polymorphic. The glutamate-oxaloacetate transaminase (AAT, E.C. 2.6.1.1) pattern in the flax species consists of four to six bands (Fig. 3). AAT synthesis in fiber flax is controlled by three loci: GOT-1, GOT-2, and GOT-3 [14]. Our results have shown that the presence of three fast bands of this enzyme (Rf 0.55, 0.56, 057) in fiber flax, intermediate flax, L. bienne, L. perenne, and L. austriacum is explained by the activity of the GOT-1 locus. In L. grandiflorum, only one intensely stained band (Rf 0.57) was detected in this part of the pattern. This polymorphism suggests that the GOT-1 locus has at least three alleles: Got-1, controlling the three active fractions, and RUSSIAN JOURNAL OF GENETICS
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got-1, controlling synthesis of one isozyme. In the middle of the enzyme pattern, two fractions were detected in the five flax species; in L. grandiflorum, these fractions were minor (Fig. 3). This suggests that variability of these isozymes may be related to interspecific differences in GOT-2 expression. The presence of one AAT fraction in this band in L. perenne indicates that GOT-2 has several alleles. One of these, Got-2, exhibits different degrees of dominance. Its presence in the flax genome produces two intensely stained (L. bienne, L. austriacum, L. usitatissimum convar. elongatum, L. usitatissimum convar. usitatissimum) or minor (L. grandiflorum) isozymes. The other allele (got-2) is recessive. Plants carrying got-2 (L. perenne) exhibit one fraction of the enzyme. The activity of locus GOT-3 controlling mitochondrial isoforms [9] is low at the blossoming stage: only one band (Rf 0.54) is detected on the zymograms (Fig. 3). In L. perenne and intermediate flax, GOT-3 is inactivated, since the AAT pattern lacks isozymes controlled by this locus. These data indicate that loci GOT-1 and GOT-2 are polymorphic, and GOT-3 is monomorphic. Zymograms of cytochrome-c-oxidase (CO, E.C. 1.9.3.1) of the flax species studied include three to nine fractions (Fig. 4a). The CO isozyme polymorphism manifested in both altered staining intensity and elec2005
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trophoretic mobility of particular components and their complete disappearance. The formation of the isoforms of this enzymes was shown to be controlled by three loci: CYT-1, CYT-2, and CYT-3. The complete CO pattern in the leaves of fiber flax at the stage of etiolated seedlings consists of nine bands (Fig. 4b) [14]. At the blossoming phase, zymograms of fiber flax (cultivar Svetoch) and L. bienne displayed seven components (Fig. 4a). The absence of isoforms with Rf 0.20 and Rf 0.04, as well as the minority of the Rf 0.05 fraction, showed that locus CYT-2 in these species has allele cyt-2 [14]. In L. austriacum and intermediate flax, six CO fractions were revealed. The absence of three bands (Rf 0.04, 0.05, and 0.20) on zymograms of these flax species suggests that locus CYT-2 in them is inactivated. Nine-band CO pattern was characteristic of L. perenne. The presence of isoforms with Rf 0.04, 0.05, and 0.20 in this species indicates the Cyt-2 allele in its genome. As in most of the flax species examined, CO pattern of L. perenne had components with Rf 0.06, 0.15, and 0.67. According to [14], synthesis of these isozymes is controlled by the CYT-1 locus. However, the activity of the Rf 0.67 fraction in L. perenne is reduced, judging by its gel staining intensity. The same CO isoform activity was found in L. austriacum. Linum grandiflorum lacked components with Rf 0.67 and Rf 0.06. This may indicate that the CYT-1 locus is polymorphic and multiallelic. Three intense bands were found in the middle of the patterns of most of the flax species examined (Fig. 4a). Their electrophoretic mobilities in fiber flax, intermediate flax, L. bienne, and L. austriacum were 0.27, 0.33, and 0.37, respectively. In L. perenne, three of these components showed higher mobilities (Rf 0.41, Rf 0.44) than the third one (Rf 0.27). In L. grandiflorum, only two bands were found in this zone (Rf 0.27 and 0.33). These data suggest that locus CYT-3 controlling these isoforms is polymorphic. Analysis of the results showed that flax loci CYT-1, CYT-2, and CYT-3, controlling CO isozymes, are polymorphic. The fatty acid content in plant oils shows the metabolic status of dormant seeds. Estimation of this parameter may serve as a reliable “fingerprinting” for chemotaxonomy of gymnosperms [15]. The amount and proportions of various fatty acids in oil are genetically determined but depend on the duration of seed storage and conditions of plant cultivation. The major fatty acids of the Linum oil are palmitic (C16:0; hexadecanoic), stearic (C18:0; octadecanoic), oleic (C18:1 (n-9); cis-9-octadecanoic), linoleic (C18:2 (n-6); linoleic), and α-linolenic (C18:3(n-3); cis/trans-linoleic) [16]. Comparative analysis of the absolute and relative content of fatty acids in the seed oil of the flax species examined revealed considerable interspecific heterogeneity in these values (table). A specific feature of L. grandiflorum seed oil is the maximum content of palmitic, stearic, and oleic acids, and the minimum content of linoleic and α-linolenic acids. In L. perenne, the reverse trend in the relative proportion of these components was found. Average amounts of the major
fatty acids were characteristic of fiber flax. A comparison of the iodine values, which reflect concentrations of mono- and polyunsaturated components in the total pool of fatty acids permitted isolation of L. bienne and intermediate flax as flax species having maximum values of this parameter. In addition to the major fatty acids, seed oil of the flax species examined contained the following minor ones: capric (C10:0; n-decanoic), lauric (C12:0, dodecanoic), myristic (C14:0; tetradecanoic), myristoleic (C14:1; cis-9-tetradecanoic), pentadecanoic (C15:0; pentadecanoic), palmitoleic (C16:1 (n-9); cis-9-hexadecenoic), elaidic (C18:1 (n-9); trans-9-octadecenoic), arachidic (C20:0; n-eicosanoic), eicosenoic (C20:1 (n-9); eicosenoic), eicosadienoic (C20:2 (n-6); cis-11,14eicosadienoic), eicosatrienoic (C20:3 (n-3); cis-11, 14, 17-eicosatrienoic), behenic (C22:0; docosanoic), and erucic (C22:1 (n-9): cis-13-docosenoic) (table). Note that some of these acids—lauric, eicosadienoic, eicosatrienoic, and erucic—were not found in all of the flax species. For instance, erucic acid was detected only in L. austriacum and L. perenne. According to some authors, fatty acid composition is significant for understanding the evolution of flax species [5]. It was found that a high content of α-linolenic acid in flax seed oil correlates with less complex genetic system, indicating a more primitive type. Our results have shown that the minimum amount of this acid was characteristic of seeds in L. grandiflorum and L. austriacum, which are the most primitive of the flax species studied. The results presented in this study indicate genetic diversity of the flax species in isozyme patterns of 6phosphogluconate dehydrogenase, glutamate-oxaloacetate transaminase, and cytochrome-c-oxidase in leaves, and the content and the relative amounts of fatty acids in seed oil. The comparative analysis of isozyme patterns showed that the loci controlling the isozyme synthesis are polymorphic. The loci controlling 6-phosphogluconate dehydrogenase (PGD-1, PGD-2) and cytochrome-c-oxidase (CYT-1, CYT-2, CYT-3) were found to be polymorphic. Two of the loci controlling glutamate-oxaloacetate transaminase (GOT-1 and GOT-2) are also polymorphic, and the third one (GOT-3) determining the formation of mitochondrial AAT forms, is monomorphic. Each of the flax species studied was characterized by strictly defined amounts and proportions of fatty acids, i.e., the fatty acid composition of seed oil may be used as a species-specific trait. These data also suggest that genetic polymorphism is a result of selection pressure on the integral metabolic phenotype. The revealed similarity of the flax species in the enzyme patterns may indicate their close phylogenetic relationships. These results are supported by studies conducted with the use of seed protein electrophoresis and RAPD–PCR [2, 17], suggesting divergence of these species from the common ancestor. Isozyme pat-
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terns in L. grandiflorum were substantially different from those of the other species examined. Comparative studies of flax genomes by other methods also showed that L. grandiflorum was different from other flax species [1, 2, 4, 17]. The differences detected on the zymograms indicate that L. grandiflorum is a phylogenetic branch diverged at an early stage of the flax evolution. REFERENCES 1. Muravenko, O.V., Samatadze, T.E., Popov, K.V., et al., Comparative Genome Analysis in Two Flax Species by C-Banding Patterns, Rus. J. Genet., 2001, vol. 37, no. 3, pp. 253–256. 2. Kutuzova, S.N., Gavrilyuk, I.P., and Eggi, E.E., Prospects of Using Protein Markers for Improving the Systematics and Elucidating the Evolution of the Genus Linum L., Tr. Prikl. Bot. Genet. Sel., 1999, vol. 156, pp. 29–39. 3. Meyer, S.E. and Kitchen, S.G., Life History Variation in Blue Flax (Linum perenne: Linaceae): Seed Germination Phenology, Am. J. Bot., 1994, vol. 81, no. 5, pp. 528– 535. 4. Lemesh, V.A., Malyshev, S.V., and Khotyljova, L.V., Use of Molecular Markers to Study Genetic Diversity in Flax, Dokl. Nats. Akad. Nauk Belarusi, 1999, vol. 43, no. 3, pp. 70–72. 5. Polyakov, A.V., Biotekhnologiya v selektsii l’na (Biotechnology in Flax Breeding), Tver, 2000. 6. Glazko, V.I. and Sozinov, I.A., Genetika izofermentov zhivotnykh i rastenii (Genetics of Animal and Plant Isozymes), Kiev: Urozhai, 1993. 7. Diederichsen, A., Comparison of Genetic Diversity of Flax (Linum usitatissimum L.) between Canadian Cultivars and World Collection, Plant Breed., 2001, vol. 120, pp. 360–362.
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8. Davis, B.J., Disc Electrophoresis: II. Method and Application to Human Serum Protein, Ann. N. Y. Acad. Sci., 1964, vol. 121, no. 3, pp. 402–427. 9. Yurenkova, S.I., Titok, V.V., and Khotyljova, L.V., Ontogenetic Polymorphism of Isozyme Systems in Swedish Flax, Dokl. Nats. Akad. Nauk Belarusi, 2001, vol. 45, no. 1, pp. 79–82. 10. Welch, R.W., A Micro-Method for the Estimation of Oil Content and Composition in Seed Crops, J. Sci. Food Agr., 1977, vol. 28, no. 6, pp. 635–638. 11. Haas, M.J., Bloomer, S., and Scott, K., Simple, HighEfficiency Synthesis of Fatty Acid Methyl Esters from Soapstock, J. Am. Oil Chem. Soc., 2000, vol. 77, no. 4, pp. 373–379. 12. AOCS Recommended Practice Cd 1c-85. Calculated Iodine Value, Official Methods and Recommended Practices of the American Oil Chemist’s Society, Firestone, D., Ed., Champaign, IL: AOSC, 1998, 5th ed. 13. Yurenkova, S.I. and Khotyljova, L.V., Estimation of Genetic Diversity of Swedish Flax by Isozyme Spectra, Dokl. Nats. Akad. Nauk Belarusi, 2001, vol. 45, no. 4, pp. 72–75. 14. Yurenkova, S.I., Isozyme Analysis of Genetic Polymorphism in Swedish Flax Linum usitatissimum L., Vestsi Nats. Akad. Navuk Belarusi, Ser. Biyal. Navuk, 2003, no. 1, pp. 35–40. 15. Tsevegsüren, N., Aiyzetmüller, K., Brühl. L., et al., Seed Oil Fatty Acids of Mongolian Compositae: The TransFatty Acids of Heteropappus hispidus, Asterothamnus centrali-asiaticus and Artemisia palustri, J. High Resolut. Chromatogr., 2000, vol. 23, no. 5, pp. 360–366. 16. Seed Oil Fatty Acids: SOFA Database Retrieval, http://www.bagkf.de/sofa/. 17. Lemesh, V.A. and Khotyljeva, L.V., Phylogenetic Relationships among Varieties of Cultivated Flax and Its Wild Relatives, in Biodiversity and Dynamics of Ecosystems in North Eurasia, 2000, vol. 1, pp. 70–72.
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